Heterologous Expression and Efficient Secretion of Chitosanase from Microbacterium sp. in Escherichia coli

Abstract

A recombinant expression vector, pCT7-CHISP6H, was constructed for the secretory expression of mature peptide of chitosanase (mMschito) from Microbacterium sp. OU01. The vector contains several elements, including T7 promoter, signal peptide sequence of mschito, 6 9 His-tag sequence and PmaCI restriction enzyme cloning site. In pCT7-CHISP6H, mMschito was fused into signal peptide sequence of mschito gene to construct recombinant plasmid pCT7-CHISP6H-mMschito. The recombinant plasmid was transformed into Escherichia coli BL21(DE3) and then expressed. The recombinant protein was secreted into the Luria-Bertani broth and the chitosanase activity in supernatant of the culture could reach up to 67.56 U/mL. The rmMschito in the broth supernatant was purified using HisTrap TM FF Crude column and the purified rmMschito was shown to be apparent homogeneity by 12 % SDS-PAGE analysis. Detected by 4700 MALDI-TOF-TOF-MS, the molecular weight of the purified rmMschito was 26,758.1875 and it was consistent with the predicted molecular weight. Chitosan (degree of deacetylation of 99 %) was mostly hydrolyzed into chitopentaose, chitotriose, and chitobiose by the purified rmMschito.

Improved extracellular expression and high-cell-density fed-batch fermentation of chitosanase from Aspergillus Fumigatus in Escherichia coli

“…OU01 and Bacillus sp, respectively, were also expressed in E. coli through secretion under the direction of MSCHITO and PelB signal peptides. However, the extracellular activity were only 67.56 U/mL in shake flask [12] and 186 U/mL in 7-L fermenter [13]. Therefore, the active CSN expressed in E. coli through our optimized method reported the highest activity so far.…”

“…For example, two chitosanasegenes that originated from Microbacterium sp. OU01 and Bacillus sp, respectively, were expressed in E. coli with the extracellular activity of only 67.56 U/mL in shake flask [12] and 186 U/mL in 7-L fermenter [13]. Contrarily, a chitosanase derived from Bacillus subtilis HD145 was expressed in P. pastoris with relative high activity of 9000 U/mg [14].…”

Genomics and transcriptomics reveal new molecular mechanism of vibriosis resistance in fish

“…After treatment with methanol at the final concentration of 1% for 4 days, the cells were pelleted out from the culture medium by centrifugation at 8,000 r/min for 10 min at 4 °C. The supernatant was used to purify the recombinant Chst12 by affinity chromatography using Ni-NTA-agarose resin ( 49 ). The purified protein was identified by 12% SDS-PAGE.…”

An extracellular Cu/Zn superoxide dismutase from Neocaridina denticulata sinensis: cDNA cloning, mRNA expression and characterizations of recombinant protein

“…Based on our previously constructed secretory vector pCT7-CHISP6H [34], a pair of primers pCT7-CHISP6H-Nd-ecCu/Zn-SOD-F/R (Table 1) was designed for PCR ampli cation and sequencing plasmid pMD19-Nd-ecCu/Zn-SOD was used as the template. The products were recovered and ligated into pCT7-CHISP6H using ABclonal MultiF Seamless Assembly Mix (ABclonal ® Technology, Wuhan) at 50 °C for 30 min.…”